ABSTRACT The proposal investigates transcription factors encoded by the ETS gene family. These regulatory proteins play central roles in a variety of human cancers, including prostate cancer, Ewings sarcoma and various leukemias. DNA binding transcription factors, like those in the ETS family, orchestrate regulation of gene expression by binding to sequence motifs within promoter and enhancer elements and by recruiting additional multi-protein complexes. This important regulatory process is complicated by the conservation of DNA binding properties among related transcription factors. This proposal addresses how the ETS factors find functional sites in the genome and mediate the assembly of both positive- and negative-acting machinery. We employ a combination of biochemical and genomic approaches including NMR-based structure determination and genome-wide occupancy. Aim 1 will determine the mechanism of autoinhibition of DNA binding in ETS factors and how this on-board repressive activity is used in biological regulation. We will determine the mechanism of ETV6 autoinhibition and how it plays a role in the repression of transcriptional targets. Based on our findings with ETS1 and ETV6, we will next test for autoinhibition of oncogenic ETS factors, ERG and ETV1, and decipher possible regulatory routes for DNA binding. Aim 2 focuses on the PNT domain, which represents a divergence point in the ETS family serving to individualize family members. We will investigate the three PNT domain-binding factors for the oncogenic ETS factor ERG that implicates this single factor in both negative and positive transcriptional regulation. Aim 3 investigates the redundant and specific use of the ETS family within cell-specific networks. ETS factors have a redundant function at the proximal promoters of housekeeping genes, yet can display specific properties at enhancers. We will disrupt ETS networks in normal and diseased cell states to decipher rules for redundant versus specific occupancy. We will test for mechanistic roles of ETS factors in regulating chromatin dynamics and DNA methylation with a focus on redundantly-occupied promoter proximal sites. Our investigation on mechanistic aspects of ETS factors will guide future interventional strategies in the cancer setting. Furthermore, our identification of downstream transcriptional targets of oncogenic ETS factors can help elucidate mechanisms of carcinogenesis.